Process and device for separating biological particles contained in a fluid by means of filtration

ABSTRACT

The invention relates to a method of separating biological particles from the liquid containing same for purification, analysis and optionally diagnostic purposes. The inventive method comprises at least one step involving vertical filtration through a filter having a porosity that is adapted to the type of biological particles to be separated, such that said particles are retained by the filter. The invention is characterised in that: (i) the method involves the use of a filter comprising at least one basic filtration zone, whereby each basic filtration zone has a limited surface area; and (ii) the surface area of each basic filtration zone and the number of basic filtration zones are selected as a function of the type of liquid to be filtered, the type of biological particles to be separated and the volume of liquid to be filtered.

This application is a Divisional of 14/517,288, filed Oct. 17, 2014,which is continuation of application Ser. No. 11/908,545, filed Sep. 13,2007, which is the National Stage of International Application No.PCT/FR2006/000562, filed Mar. 14, 2006, (which is hereby incorporated byreference).

The present invention relates to the separation of biological particlescontained in a fluid, which can be separated on the basis of their size,separation being carried out by vertical filtration using a filtersuited to the nature of the particles to be isolated. The particles areisolated in particular for the purposes of purification or analysis andpossibly for diagnosis.

To develop non-invasive diagnostic methods, particularly in the prenatalfield, or early diagnosis methods, particularly in the field ofoncology, it has been proposed in patent applications FR 2 782 730 andFR 2 824 144 to seek rare characteristic cells in biological fluidstaken from patients. In these methods, the cells to be detected aresearched for by means of an enrichment operation by filtration using inparticular an ISET-type filtration machine such as the machine describedin patent WO 91/11245. These methods consist of filtering a biologicalfluid, which may have undergone specific preparation, through a filterthe pores or porosity of which are suitable, in such a way that thecells sought form on the filter a filtration residue, which is thenanalysed. The porosity of the filter, the preparation of the biologicalfluid and the analysis method are chosen according to the nature of thecells sought.

These methods were developed initially using a filtration appliancedesigned to search for particles in milk for quality control purposes.Although necessary adaptations were made, given the nature of thefiltered fluids and the particles sought, these methods still havedrawbacks. In particular, blood is difficult to filter.

In about 30% of cases, filtration of this fluid is interrupted byclogging, making it unusable.

In addition, the device used to contain the sample of fluid to befiltered comprises a mechanism to achieve a seal that is ratherinconvenient to use.

Finally, and in general, filtration reliability sufficient for useexperimentally in a laboratory is not sufficient for se for massdiagnostic purposes.

The object of the invention is to overcome this drawback by offering ameans of making the operation of isolating by vertical filtration thecells contained in a fluid or more generally of separating biologicalparticles from a fluid that contains them more reliable so as to make itsuitable in particular for use for diagnostic purposes.

Accordingly, the invention relates to a process for separatingbiological particles and the fluid that contains them for the purposesof purification or analysis and possibly for diagnosis, comprising atleast one vertical filtration stage through a filter the porosity ofwhich is suited to the nature of the biological particles to beseparated so that said biological particles are retained by the filter,characterised in that a filter is used comprising at least oneelementary filtration area, each elementary filtration area having alimited surface, and in that the surface of each elementary filtrationarea and the number of elementary filtration areas is chosen accordingto the nature of the fluid to be filtered, the nature of the biologicalparticles to be separated and the volume of fluid to be filtered.

Each elementary filtration area of said process has a surface equal tothat of a disk with a diameter of between 0.6 cm and 3 cm, and thenumber of elementary filtration area is chosen so that the ratio of thevolume of fluid filtered to the filtration surface is less than 40ml/cm², and preferably greater than 0.14 ml/cm².

Preferably, each elementary filtration area has a surface equal to thatof a disk with a diameter greater than or equal to 0.8 cm.

Preferably, the filter has pores calibrated to a size of between 3 μmand 100 μm and a pore density of between 3×10³ and 5×10⁶ pores/cm².

Preferably, filtration is carried out by a reduction in pressure ofbetween 0.05 bar and 1 bar with, possibly, an increase in pressure ofless than 1 bar.

To carry out filtration, it is preferable to use a filter forming abadge suitable to be associated with a means of analysing filtrationresidues by locating the elementary filtration areas.

Preferably, the badge forming the filter is incorporated in a single-usefiltration module comprising at least one chamber for containing thefluid to be filtered, and that can be treated before use to sterilise itor to free it from enzymes that digest DNA, RNA or proteins.

The biological particles to be separated are, for example, cells. Inthis case, prior to filtering the fluid containing the cells, a sampleof fluid for filtering may be prepared from a sample of fluid containingcells such as a biological fluid or cell culture by pre-enriching itwith the cells to be separated and/or by diluting it.

The fluid containing the cells may be blood and, preferably, the filterin this case has calibrated pores of between 5 μm and 25 μm.

The biological particles may also be fibrins.

The fluid containing the biological particles is urine and thecalibrated pores of the filter are between 8 μm and 100 μm.

The process can be used for the detection of cells for diagnosticpurposes such as tumour, foetal, endothelial, fibroblastic, muscle,nerve or monocytal cells, cell strains, organ cells, precursors orhaematopoietic cells, in a biological fluid such as blood, urine,ascites, cephalorachidian fluid, milk, pleural extravasation, fluid forwashing the neck of the uterus, cell suspension fluid obtained bybiopsy, by a surgical method or by mouth washing, or for the detectionof animal or vegetable cells.

The invention also relates to a filtration module for implementing theprocess, said module comprising:

-   -   a chamber block comprising at least one compartment closed at        its lower portion by a base comprising at least one opening;    -   a filter support drawer comprising at least one hole, each hole        being arranged facing an opening in the chamber block;    -   a filter gripped between the lower face of the chamber block and        the support drawer.

In this module, the dimensions of each opening in the base of thechamber block and the dimensions of each hole in the filter supportdrawer are such that each pair made up of an opening in the base of thechamber block and the associated hole in the filter support draw, definean elementary filtration area of limited surface and in that the usefulvolume of each compartment is proportional to the number of elementaryfiltration areas situated in the base of the compartment.

Preferably, the surface of an elementary filtration area is equal tothat of a disk with an equivalent diameter of between 0.6 cm and 3 cm,and the ratio of the useful volume of each compartment to the sum of thesurfaces of the openings comprised in the base of the compartment isless than 40 ml/ cm², and preferably greater than 0.14 ml/cm².

Preferably, the dimensions of at least one opening in the base of thechamber block and of a corresponding hole in the filter support drawerare such that the surface of the corresponding elementary filtrationarea is greater than or equal to that of a disk 0.8 cm in diameter.

Preferably, at least one compartment may be divided into partcompartments by at least one removable separation wall, such that atleast one part compartment comprises in its base at least one openingand that the ratio of the volume of said part compartment to the sum ofthe surfaces of the openings in the base of the part compartment is lessthan 40 ml/cm², and preferably greater than 0.14 ml/cm².

Preferably, the filtration module comprises a grooved sealing jointarranged between the base of the chamber block and the filter,comprising at least one hole corresponding to a hole in the base of thechamber block, the hole being surrounded by at least one projecting lip.

In addition, the filtration module preferably also comprises a platejoint between the filter and the filter support, comprising at least oneopening opposite a hole in the filter support.

The filter may form a badge the central portion of which comprises atleast one porous area and the periphery of which forms a framecomprising means for indexing its position on the filter support.

The indexation means are, for example, at least two holes of differentdiameter designed to cooperate with studs of corresponding diameterprovided on the filter support.

Preferably, at least a central porous portion of the filter comprisesbetween 3×10³ and 5×10⁶ pores per cm² of between 3 μm and 100 μm.

Preferably, the filtration module also comprises at least one stopperfor closing the upper opening of a compartment.

Preferably, the chamber block comprises, at its lower portion, a rimextending outwards and cooperating with at least one assembly pinallowing the filter to be gripped between the filter support and thechamber block, the assembly pin comprising a breakable end extendingabove the rim of the chamber block.

Preferably, all its parts are made of materials suited to asterilisation operation or designed to render them free from RNases,DNases or proteinases.

Finally, the invention relates to a filtration module support forretaining a filtration module on a filtration machine, comprising atleast one cam that can move between an open position and a grippingposition, designed to put pressure on the filter between the filtersupport and the chamber block.

Preferably, at least one cam is designed so that, if the filtrationmodule comprises at least one fixing pin one end of which is breakable,the end of at least one fixing pin is cut when pressure is applied tothe filter by at least one cam.

The support block forms part of a filtration machine.

Preferably, the filtration module also comprises a means designed tocooperate with a complementary means on a support block, so as to imposethe orientation of the filtration module in relation to the supportblock, and the support block comprises a means designed to cooperatewith a means on a filtration module, so as to index the orientation ofthe filtration module in relation to the support block.

The invention will now be described in more detail but in a non-limitingmanner with regard to the accompanying drawings in which:

FIG. 1 is a perspective view of a filtration module associated with afiltration module support block;

FIG. 2 is an exploded perspective view of a filtration module;

FIGS. 3A and 3B are two views in cross-section of a chamber block of afiltration module;

FIG. 4 is a perspective view with cross-section of a grooved sealingjoint;

FIGS. 5A and 5B are perspective views, in particular with a crosssection, of a filter support drawer;

FIG. 6A is a perspective view of a filtration module arranged on asupport block;

FIG. 6B is an enlarged perspective view of means for gripping the baseof a filtration module arranged on a support block of the filtrationmodule; and

FIG. 7 is a separated perspective view of a filtration module after use,in which the chamber block is separated from the filter support drawercomprising a filter.

FIG. 8 is a perspective view of a filtration module according to avariant.

FIG. 9 is a perspective view of a badge-filter.

The method for isolating biological particles contained in a fluid,according to the invention, consists of filtering the fluid on a filterwith characteristics suited to the nature of the particles to beisolated. The biological particles may be cells, red blood cells,platelet aggregates, fibrins or tissue waste. The filtered fluid is inparticular a fluid obtained from a sample of biological fluid that mayhave undergone prior treatment to facilitate the isolation by filteringoperation. This prior operation, which will be described in more detaillater, comprises in general, particularly when the particles to beisolated are cells, one or a plurality of the following operations:chemical treatment designed to pre-enrich the cell to be isolated,dilution, chemical treatment designed to facilitate separation byfiltration of the cells to be isolated.

As well as these conditions for preparing samples of fluid forfiltering, the inventors noted that to achieve good reliability in theprocess of isolating cells to be detected, it was necessary to adaptcertain characteristics of the filter to the volume of fluid filtered.In particular, the filter must be divided into elementary filtrationarea each having a surface equal to that of a disk of diameter ofbetween 0.6 cm and 3 cm, and preferably greater than 0.8 cm and evenbetter between 0.8 cm and 1.5 cm. The elementary filtration areas may bein the shape of a disk, for example.

In addition, the quantity of fluid to be filtered, which must passthrough each of the elementary filtration areas, must be between 1 mland 100 ml, and preferably this volume should be between 8 ml and 15 ml.

Thus, to filter a particular sample a device must be used to define anumber of elementary filtration areas on the filter in proportion to thevolume of the sample to be filtered.

In general, the volume of the sample to be filtered depends on the onehand on the volume of biological fluid that could be taken initially,and on the other hand on a possible dilution which depends in particularon the nature of the biological particles to be separated. The volumetaken depends in particular on the nature of the fluid taken and the ageof the patient from whom the fluid is taken. A person skilled in the artknows how to determine the volumes to be taken depending on the natureof the fluid taken and on the patient from whom it is taken.

Dilution depends in particular on the number of particles per unit ofvolume that can be found in the fluid taken. Indeed, if filtration is tobe carried out under satisfactory conditions, the number of particles tobe isolated per unit of volume of fluid to be filtered should not be toogreat to avoid clogging the filter. Moreover, if the process is intendedto detect particular rare cells mixed with a far greater number ofcells, the number of cells per unit of volume should not be too small,so as to achieve a reasonable probability of finding the cells sought onthe filter. A person skilled in the art also knows how to determinethese dilution rates depending on the nature of the fluid in questionand the type of cell sought.

The biological sample taken from a patient may, for example, be blood,urine, ascites, cephalorachidian fluid, milk or pleural extravasation;it may also be fluid from washing the neck of the uterus or any otherfluid that may result from taking a biological sample from a patient.

The analysis method may also be used to search for cells in samples thathave not been taken directly from patients, and for example, in samplestaken in cell culture mediums made from smears or biopsies or from humanor animal tissue samples or, further, in human or animal cell lineculture mediums.

If the biological fluid taken is blood, the amount taken is generallybetween 1 ml and 20 ml, and the blood is diluted by a ratio that variesfrom 1 in 5 to 1 in 20 to obtain a sample of fluid for filtering which,in these conditions, is filtered over one to 20 elementary filtrationareas.

For all other fluids, the samples are approximately 5 ml to 10 ml andare diluted in a ratio of between 1 in 2 and 1 in 10, or they may not bediluted. These samples are filtered over a number of elementaryfiltration areas which may be as many as 5 or even more, particularly ifit is a 10 ml sample that has been diluted in a ratio of 1 in 10.

The cells that may be sought are in particular tumour cells, foetalcells, endothelial cells, fibroblastic cells, muscle cells, nerve cells,monocytal cells, cell strains, organ cells (hepatic, renal, etc . . . ),precursors and haematopoietic cells. This list, which is given as anexample, is not limitative.

Before filtration, the cells may be pre-enriched by treatment of thedensity gradient type or by lysis of cells that are of no interest, orby immunomediated methods, by positive or negative screening, bystimulating the cells sought to proliferate, etc.

This list is not limitative, and a person skilled in the art knows howto choose a pre-enrichment process suited to the nature of the cellsthat he or she seeks to isolate.

As well as the pre-enrichment treatment, the fluid sample containingcells may be treated by a reagent according to the nature of the cellssought, to facilitate the separation by filtration operation.

The aim of the treatment may be to lyse red blood cells andanticoagulate the blood if the biological sample contains blood, andconsists, for example, of adding saponin and EDTA.

The aim of the treatment may also be to fix nucleated cells, for exampleby the addition of formaldehyde, if the filtration is intended toisolate fixed cells. In this case, the object of the treatment is tomake enrichment possible.

If the filtration is intended to isolate non-fixed cells, the biologicalsample may be treated with a reagent and under conditions suitable fortemporarily rendering biological membranes rigid (for example, by theaddition of polysaccharide, DMSO, by cold, etc.).

A person skilled in the art knows how to choose the most suitablemethod, according to the nature of the cells sought.

The biological sample which may have been diluted, pre-enriched ortreated with a reagent to allow filtration suited to the end sought, isthen filtered through a filter made of polycarbonate or an equivalentmaterial that has calibrated pores of a size between 1 μm and 100 μm andsuited to the nature of the particles to be separated. This size ispreferably between 3 μm and 25 μm, and is about 8 μm, for example,particularly if tumour cells or epithelial cells are to be isolated.

Pore density is suited to the nature of the particles to be separated.Preferably, the pore density of the filter is between 5×10³ and 5×10⁶pores/cm² and even better between 5×10⁴ and 5×10⁵ pores/cm².

Filtration is performed preferably be a reduction in pressure of between0.05 bar and 1 bar, and preferably of approximately 0.1 bar. Filtrationmay be assisted by a slight increase in pressure on the fluid situatedabove the filter. This increase in pressure must however be less than 1bar. These conditions are particularly suited to cell separation.

The process may be used for different objectives, for example to searchfor rare cells in suspension in a biological fluid, so as to allowdiagnosis or to purify a fluid to allow analysis in good conditions ofthe elements in solution.

If the process is used to search for cells and analyse them, afterfiltration, the filter that has been used to filter the fluid isrecovered ensuring that the filtration areas are clearly identified andthat a link can be made between these filtration areas and the samplethat was filtered. The filter is then used to analyse the cells that itmay have been possible to recover in the filtration areas.

These analysis methods, which are known per se, are for example of thefollowing types: cytological staining (haematoxylin, eosin, etc.),immunomarking (immunohistochemistry, immunofluorescence) PNA, FISH,PRINS, PCR in situ or other molecular technique, spectrophotometry,laser microdissection followed by targeted molecular analyses on the DNA(DNA extraction, genotyping, quantitative PCR, mutation analysis, CGH(comparative genomic hybridisation)) RNA (extraction and analysis by PCRof transcripts, quantitative PCR) and proteins (protein extraction,microsequencing, etc.).

The molecular analyses may be performed on enriched cells held on thefilter and transposed onto a slide by a technique similar to theSouthern technique, individually micro-dissected from the filter or fromthe slide according to defined criteria (morphological characteristicsof the cells with or without marking of different natures) and subjectedto individual or pooled molecular analysis.

The cells may also be detached from the filter by washing with anappropriate buffer to extract and analyse their DNA, RNA and proteins.

The elements isolated by filtration are then examined with a microscopeand analysis of the images obtained on the filter may be carried outmanually or by automated means, in particular by using image analysisequipment.

The process may also be used to purify a biological fluid such as urinecontaining in solution the DNA, RNA or proteins that are to be analysed.The purpose of purifying the fluid is to eliminate all the biologicalparticles present in the fluid, which could interfere with the analysis.In this case, the filters are not kept and it is the filtered fluid thatis analysed.

This filtration method and the sample preparation and analysis methodsmay be used as stated previously in particular for the purpose ofdiagnosis to detect pathologies associated with the presence ofparticular cells possibly in extremely small quantities. In particular,the process can be used to detect cancerous cells that may have beenreleased into a patient's blood during a surgical operation. A personskilled in the art knows what cells can be searched for to detect aparticular pathology.

To ensure that these analyses are carried out under conditions ofsatisfactory reliability, particularly in the context of hospitals ormedical analysis laboratories, it is desirable that the appliances usedshould provide good reproducibility and reliability of the conditions inwhich the analysis was performed. Accordingly, the inventors havedeveloped devices suitable for performing these analyses in conditionsof satisfactory reliability.

These devices will now be described.

To carry out the analyses in conditions of acceptable reliability, thefluids to be filtered are collected in a filtration module, preferably asingle-use filtration module, referenced generally as 1 in FIG. 1 anddesigned to be mounted on a support block referenced generally as 2,forming part of the filtration machine (which is not illustrated in itsentirety in the figure).

The filtration module 1 is placed on the support block to perform thefiltration. When filtration has finished, the filtration module 1 isremoved from the support block 2 and the filter contained in thefiltration module is removed so that the analyses can be performed aswill be explained later.

The filtration module 1, illustrated in exploded form in FIG. 2,consists of a stack comprising, from top to bottom, a chamber block 11,a grooved joint 12, a filter 13, a plate joint 14 and a filter supportdrawer 15.

The filter 13 is laid flat against the lower face 110 of the chamberblock 11 by means of the filter support drawer 15, the seal between thefilter 13 and the chamber block 11 being provided by the grooved joint12, and the seal between the filter 13 and the filter support drawer 15being provided by the plate joint 14.

When the assembly is fitted together, the filter support drawer is heldin position against the chamber block 11 by means of two assembly pins16 which pass through holes 151 in the filter support drawer and hookinto holes 112 provided in a lower rim 111 of the chamber block 11,extending outwards.

The chamber block 11, made of plastics material, comprises a body 114divided into two compartments 113 open at their upper portion 115 andclosed at their lower portion by a base 116 comprising a plurality ofcircular openings 117 with a diameter of between 0.6 cm and 2 cm. Thebase also comprises a rim 111 forming a small collar in which openings112 designed to receive the hooks of the assembly pins 16 are providedon the two side portions of the chamber block. The compartments 113 maybe closed at their upper portion by removable stoppers 17. The filletradii of the base and walls of each compartment are rounded so as not tocreate an area where particles to be filtered can be trapped.

As can be seen in FIG. 3B, the compartments 113 may comprise verticalgrooves 118 designed to receive separation slides 119 allowing thecompartments 113 to be divided into part compartments of smaller volume.The grooves 118 are arranged such that any separation slides 119 arealways arranged between two circular openings 117.

To ensure appropriate filtration, the volume of the compartments is inproportion to the number of circular openings 117, such that the totalvolume of the compartment or more precisely the maximum quantity offluid that the compartment can receive is between 0.14 ml/cm² and 40ml/cm² multiplied by the sum of the surfaces of the openings comprisedin the base of the compartment. In particular, the height of thecompartment and the cross-section of the base are such that not only arethese conditions respected for a complete compartment but also such thatthey are respected for any part compartment delimited by means of one ora plurality of removable sealed separation walls 119. In particular, ifan opening 117 is isolated by two removable walls 119, or by oneremovable wall 119 and by the wall of the compartment, so that it onlycomprises one opening 117 in the base, the volume situated above theopening 117 is suitable for receiving a maximum of 20 ml of fluid forfiltering. As an example, each compartment may have a volume of 110 mland comprise 5 openings in the base.

In a variant illustrated in FIG. 8, the chamber block 11 comprises acompartment 113, comprising five elementary filtration areas, acompartment 113A comprising one elementary filtration area and twocompartments 113B comprising two elementary filtration areas. Eachcompartment comprises a removable stopper 17, 17A, 17B. It will be notedthat with this arrangement, it is possible to choose the number ofelementary filtration area used from between 1 and 10.

On its lower face 110, the chamber block comprises two holes ofdifferent diameter (not visible on the figure) arranged diagonally,designed to cooperate with studs 153A and 153B of the filter supportdrawer 15, to ensure precise positioning and location of the position ofthe filter in relation to the base of the chamber block.

The grooved joint 12 arranged just beneath the chamber block is made ofmoulded silicon and comprises a plurality of holes 121 designed to facethe openings 117 provided in the base of the chamber block, these holes121 being surrounded by circular lips 122, and preferably by twocircular lips, to provide a good seal. The joint may also compriselongitudinal grooves 123 for separating a first series of holes from asecond series of holes, to provide separation between the holes oppositea first compartment and the holes opposite the second compartment of thechamber block. The function of this joint is to provide a perfect sealbetween each of the holes so that the fluid passing through a holecannot be mixed with the fluid passing through another hole, whichallows simultaneous filtration of two different fluids. The projectinglips 122, and possibly 123, are arranged on the face 124 of the jointdesigned to cooperate with the lower portion of the chamber block 11.The second face 125 of the joint, designed to cooperate with the filter13, is flat.

The filter 13, which forms a badge of generally rectangular shape andwhich is flat, comprises a central filtration area 131 consisting of amembrane made of microporous polycarbonate, about 100 μm thick and of aporosity appropriate to the process. This central portion is fixed usingbiological glue on a PVC frame 132 that can be used to grasp the badgeand on which reference inscriptions can be made. The frame 132 comprisesmeans for positioning and locating the orientation of the badgeconsisting of two holes 133A and 133B arranged diagonally on the badgeand having different diameters designed to cooperate with the studs 153Aand 153B of the filter support. With this arrangement of location holes133A and 133B, only one orientation is possible to arrange the badge ona support comprising complementary location studs for the holes 133A and133B. This allows the orientation of the badge to be located veryexactly on the one hand when it is arranged in the separation module andon the other hand when it is arranged on an analysis appliance such asequipment for staining or reading under a microscope.

In a variant illustrated in FIG. 9, the badge comprises two centralfiltration areas 131A, 131B parallel to each other and which maycomprise membranes of different porosity.

The plate joint 14 is a flat plate joint made of silicon comprisingcircular openings 140 corresponding to openings 121 in the grooved jointand to openings 117 provided in the base of the chamber block. It alsocomprises holes 141A and 141B of different diameter corresponding to theholes 133A and 133B of the filter.

The filter support drawer 15 is a plate made of injected plasticsmaterial comprising an upper face 150 designed to grip the filter andjoints assembly against the lower face 110 of the chamber block 11. Thisfilter support plate comprises a series of holes 152 designed to facethe corresponding holes 140 of the plate joint, 121 of the grooved jointand 117 of the chamber block.

The upper face 150 of the filter support drawer also comprises studs153A and 153B designed to cooperate with the location holes 133A and133B of the filter badge 13, with the corresponding holes 141A and 141Bof the plate joint 14 and with the centring holes provided on the lowerface 110 of the chamber block.

As can be seen in FIG. 5B, the holes 152 designed to allow the filteredfluid to pass through comprise an upper portion in the form of a funnelwhich is extended by small tubes 154 projecting on the lower face 155 ofthe filter support drawer 15. The function of the small tubes 154projecting in relation to the lower face 155 of the filter supportdrawer is to ensure that the fluid flows properly after filtration sothat no drops are formed that could moisten the lower face of the filtersupport. It will be noted that the diameter of the upper portion of theholes 152, like the diameter of the holes 140 of the plate joint and 151of the grooved joint, are substantially equal to the diameter of theholes 117 provided in the base of the chamber block, in order to delimitelementary filtration areas of corresponding diameter on the filter. Thefilter support drawer 15 also comprises holes 151 on its two side edgesdesigned to receive the assembly pins 16. These holes are of a suitableshape to allow the introduction of the assembly pins and lock the unitin position when assembling the filtration module unit.

The assembly pins 16 each comprise a head 161 surmounted by a stem 162,of smaller diameter, the end of which forms a hook 163. The length ofthe assembly pin is suitable to allow the stack made up of the filtersupport drawer, the joints and the filter to be locked when it is laidflat against the chamber block. Thus, an assembly pin is of sufficientlength to ensure that, when it is introduced into a hole 151 of thefilter support drawer, it can penetrate into the hole 112 of the lowerrim of the chamber block 11, such that the hook 163 situated at the endof the assembly pin can hook on the upper surface of the rim 111 of thechamber block, the head 161 of the assembly pin 16 cooperating with thelower face of the filter support drawer 15 so as to maintain the filtersupport drawer locked against the base of the chamber block 11.

The filtration module as just described is designed to be arranged on asupport block 2 of the filtration module, which comprises on the onehand a support face 200 comprising a central opening 201 designed toface all the openings on the lower face of a filtration block, on theother hand a lever 202 pivoting about a pin 203 designed to manoeuvretwo cams 204 (only one of which can be seen in the figure) for grippingthe lower portion of the filtration module tightly against the surface200 of the support block, and thereby to grip the chamber block 11tightly against the filter support drawer 15 during filtrationoperations.

In a preferred variant, illustrated in FIG. 8, the lower rim 111 of thechamber block and, possibly the filter support drawer 15, comprise atone end, an enlarged portion 114 designed to cooperate with an enlargedportion 203 of the runner 204 of the support block 2 designed to receivethe base of the filtration module. This arrangement allows anorientation to be imposed on the filtration module in relation to thesupport block.

When the filtration module 1 is arranged on the support block 2, thecams are facing the rim 111 of the chamber block 11, such that when thelever is in the open position, the cams leave room for the rim 111 toslide on the upper face 200 of the support block, and when the lever isin the gripping position, the cams rest firmly on the rim 111 of thechamber block 11, so as to grip the unit formed by the filtration block,the filter, the filter support and the associated joints, against theface 200 of the filtration module support.

Moreover, each cam comprises a vane 205 projecting outwards which, whenthe rim of the filtration module is tightened, cooperates with the endof the corresponding assembly pin 16, in order to break the hook sothat, when full tightening has been achieved, the hook of the assemblypin is broken. Because of this arrangement, when the filtration moduleis tightened against the support block 2 by means of the cam, the unitis held together tightly, while, after loosening the cam, since the headof the hook has been broken, the filter support block and the chamberblock are no longer integral with each other as illustrated in FIG. 7.This allows the filter to be removed and, at the same time, renders thefiltration module unusable for another analysis. The filtration moduleis therefore a single-use module.

A block that does not comprise breakable hooks can also be used. Such ablock can then be used repeatedly, which is less reliable than a singleuse, but may nevertheless have attractions in some cases.

Before any use, the filtration module is closed using the upper stoppersand sterilised. If a sample is to be filtered for analysis purposes, asterilised filtration module is taken and arranged on a support block ofa filtration machine. Using the tightening lever, the filtration moduleis locked on the filtration machine ensuring that the filter supportplate is gripped firmly against the base of the chamber block. By doingthis, if it is a single-use module, the ends of the assembly pinscomprising hooks are broken. At least one of the compartments into whichthe fluid to be filtered is introduced is then opened so that, in eachcompartment, the ratio of the amount of fluid contained to the sum ofthe surfaces of the openings of the base of the compartment is between0.14 and 40 ml/cm². Filtration is then carried out. Once the filtrationis performed, the system is unlocked by raising the tightening lever,and the filtration module is removed.

On this filtration module, the lower portion made up of the filtersupport plate is not integral with the chamber block. The unit istherefore separated and the filter is then removed and arranged on theplaten of an observation microscope, possibly after having made a numberof preparations using reagents to allow convenient observation of thecells that may have been trapped by the filter.

The rest of the device, that is to say the chamber, the filter supportand the joint, are then thrown in the dustbin so that they cannot bereused. This single-use device has the advantage of providing goodanalysis security. In fact, the same device is used only to analyse asingle sample, which avoids all risk of a sample being contaminated byearlier samples that may have been analysed using the same device.Moreover, because of the particular geometric characteristics of boththe holes arranged in the base of the chamber and of the chamber sizes,the amounts of fluid filtered by the device are suitable to ensure thatthe amounts filtered on each of the elementary filtration areas of thefilter comply with the conditions imposed by the process to achievereliable results.

1-31. (canceled)
 32. A process for separating biological particles andthe fluid that contains them comprising subjecting a fluid containingbiological particles to at least one vertical filtration stage through afilter having a porosity suited to the nature of the biologicalparticles to be separated so that said biological particles are retainedby the filter, and the fluid passes through the filter wherein thefilter comprises at least one elementary filtration area, eachelementary filtration area having a limited surface, each elementaryfiltration area and the number of elementary filtration areas are setaccording to the nature of the fluid to be filtered, the nature of thebiological particles to be separated and the volume of fluid to befiltered, each elementary filtration area has a surface equal to that ofa disk with a diameter of between 0.6 cm and 3 cm, and the number ofelementary filtration areas is set so that the ratio of the volume offluid filtered and of the filtration surface is less than 40 ml/cm². 33.The process according to claim 32, wherein each elementary filtrationarea has a surface equal to that of a disk having a diameter greaterthan or equal to 0.8 cm.
 34. The process according to claim 33, whereinthe filter has pores calibrated to a size of between 3 μm and 100 μm anda pore density of between 3×10³ and 5×10⁶ pores/cm².
 35. The processaccording to claim 32, wherein filtration is conducted by a reduction inpressure of between 0.05 bar and 1 bar, or an increase in pressure ofless than 1 bar.
 36. The process according to claim 32, wherein thefilter is in the form of a badge configured to permit analysis offiltration residues by locating the elementary filtration areas.
 37. Theprocess according to claim 36, wherein the filter is incorporated in afiltration module comprising at least one chamber configured to containthe fluid to be filtered, which is optionally sterilized or renderedfree from enzymes that digest DNA, RNA or proteins prior to use infiltration.
 38. The process according to claim 37, wherein thefiltration module is a single-use filtration module.
 39. The processaccording to claim 32, wherein the biological particles are cells. 40.The process according to claim 39, wherein the fluid containingbiological particles is a biological fluid or cell culture pre-enrichedwith the cells or is a biological fluid or cell culture that has beendiluted.
 41. The process according to claim 39, wherein the fluidcontaining the cells is blood and the filter has calibrated pores ofbetween 5 μm and 25 μm.
 42. The process according to claim 32, whereinthe fluid containing the biological particles is urine and thecalibrated pores of the filter are between 8 μm and 100 μm.
 43. Afiltration module for implementing the process according to claim 32,comprising: a chamber block comprising at least one compartment closedat its lower portion by a base comprising at least one opening; a filtersupport drawer comprising at least one hole, each hole being arrangedfacing an opening in the chamber block; a filter gripped between thelower face of the chamber block and the support drawer, wherein thedimensions of each opening in the base of the chamber block and thedimensions of each hole in the filter support drawer are such that eachpair made up of an opening in the base of the chamber block and theassociated hole in the filter support draw, define an elementaryfiltration area of limited surface, in that the useful volume of eachcompartment is proportional to the number of elementary filtration areassituated in the base of the compartment and in that the surface of theelementary filtration area is equal to that of a disk having a diameterof between 0.6 cm and 3 cm, and in that the ratio of the useful volumeof each compartment to the sum of the surfaces of the openingscomprising the base of the compartment is less than 40 ml/cm².
 44. Aprocess according to claim 32 for isolating rare characteristic cells ina biological fluid for diagnosis purposes, comprising: a) treating asample of a biological fluid for lysis of the cells that are of nointerest; b) vertical filtration of the treated sample obtained in a)through a filter comprising pores calibrated to a size of between 3 μmand 100 μm and the density of which is between 3×10³ and 5×10⁶ per cm²,said filter comprising at least one elementary filtration area, eachelementary filtration area having a surface equal to that of a disk witha diameter of between 0.6 cm and 3 cm, and the number of elementaryfiltration areas being chosen so that the ratio of the volume of thefluid to be filtered and the sum of the surfaces of the elementaryfiltration areas is between 0.14 ml/cm² and 40 ml/cm²; and, c) analysisof the cells retained on each of the elementary filtration areas of thefilter used to filter the sample.
 45. The process according to claim 44,wherein, during the step of analysis, the cells retained on the filterare transposed onto a slide.
 46. The process according to claim 44,wherein the biological fluid is blood and a) comprises lysis of redblood cells and anticoagulating the blood.
 47. The process according toclaim 46, wherein a) further comprises adding saponin and EDTA to thebiological fluid to lyse the red blood cells and anti coagulate theblood.
 48. The process according to claim 44, wherein a) furthercomprises fixing nucleated cells.
 49. The process according to claim 44,wherein, in c), the analysis of the cells retained on the filtrationareas during b) is performed by cytological staining, immunomarking,FISH, PRINS, PCR in situ, spectrophotometry, or laser microdissectionfollowed by targeted molecular analyses on the DNA, RNA or proteins. 50.The process according to claim 44, wherein, during c) the cells retainedare detached from the filter by washing with an appropriate buffer, andthen submitted to DNA, RNA or protein analyses.